Abstract:

The present invention is related to insulin derivatives having a side
chain attached either to the α-amino group of the N-terminal amino
acid residue of B chain or to an ε-amino group of a Lys residue
present in the B chain of the parent insulin molecule via an amide bond
which side chain comprises one or more residues of ethylenglycol,
propyleneglycol and/or butyleneglycol containing independently at each
termini a group selected from --NH2 and --COOH; a fatty diacid
moiety with 4 to 22 carbon atoms, at least one free carboxylic acid group
or a group which is negatively charged at neutral pH; and possible
linkers which link the individual components in the side chain together
via amide or ether bonds, said linkers optionally comprising a free
carboxylic acid group.

Claims:

1. An insulin derivatives having a side chain attached either to the
α-amino group of the N-terminal amino acid residue of B chain or to
an ε-amino group of a Lys residue present in the B chain of the
parent insulin molecule via an amide bond wherein said side chain
comprises one or more residues of ethylenglycol, propyleneglycol and/or
butyleneglycol containing independently at each termini a group selected
from --NH2 and --COOH; a fatty diacid moiety with from 4 to 22
carbon atoms, at least one free carboxylic acid group or a group which is
negatively charged at neutral pH; and possible linkers which link the
individual components in the side chain together via amide or ether
bonds, said linkers optionally comprising a free carboxylic acid group.

2. The insulin derivative according to claim 1, wherein PEG or PPG or PBG
group has from 2 to 20; from 2 to 10 or from 2 to 5 residues of
ethyleneglycol, propyleneglycol or butyleneglycol.

3. The insulin derivative according to claim 1, wherein the sidechain
comprises a single residue of ethyleneglycol.

4. The insulin derivative according to claim 1, wherein the sidechain
comprises single residues of ethylenglycol, propyleneglycol and
butyleneglycol alone or in combination.

5. The insulin derivative according to claim 4, wherein the sidechain
comprises one residue of propyleneglycol and one residue of
butyleneglycol.

6. The insulin derivative according to claim 1, wherein the fatty diacid
comprises from 4 to 22 carbon atoms in the carbon chain.

7. The insulin derivative according to claim 6, wherein the fatty diacid
comprises from 6 to 22, from 8 to 20, from 8 to 18, from 4 to 18, from 6
to 18, from 8 to 16, from 8 to 22, from 8 to 17 or from 8 to 15 carbon
atoms in the carbon chain.

8. The insulin derivative according to claim 1, wherein the linker is an
amino acid residue, a peptide chain of 2-4 amino acid residues or has the
motif α-Asp, β-Asp, α-Glu, γ-Glu, α-hGlu,
δ-gGlu, --N(CH2COOH)CH2CO--,
--N(CH2CH2COOH)CH2CH2CO--,
--N(CH2COOH)CH2CH2CO-- or
--N(CH2CH2COOH)CH2CO--

9. The insulin derivative according to claim 1, wherein the Lys residue in
the B chain of the parent insulin in either position B3, B29 or in one of
positions B23-30.

10. The insulin derivative according to claim 1 having the formula
##STR00071## wherein Ins is the parent insulin moiety which via the
α-amino group of the N-terminal amino acid residue of the B chain
or an ε-amino group of a Lys residue present in the B chain of
the insulin moiety is bound to the CO-- group in the side chain via an
amide bond;each n is independently 0, 1, 2, 3, 4, 5 or 6;Q1,
Q2, Q3, and Q4 independently of each other can be selected
from: (CH2CH2O)s--; (CH2CH2CH2O)s--;
(CH2CH2CH2CH2O)s--;
(CH2CH2OCH2CH2CH2CH2O)s-- or
(CH2CH2CH2OCH2CH2CH2CH2O)s--
where s is 1-20; --(CH2)r-- where r is an integer from 4 to 22;
or a divalent hydrocarbon chain comprising 1, 2 or 3 --CH═CH-- groups
and a number of --CH2-- groups sufficient to give a total number of
carbon atoms in the chain in the range of 4 to 22; --(CH2)t--
or --(CH2OCH2)t--, where t is an integer from 1 to 6;
--(CR1R2)q--, where R1 and R2 independently of
each other can be H, --COOH, (CH2)1-6COOH and R1 and
R2 can be different at each carbon, and q is 1-6;
--((CR3R4)q1)1--(NHCO--(CR3R4)q1--NHCO-
)1-2--((CR3R4)q1)1 or
--((CR3R4)q1)1--(CONH--(CR3R4)q1--CONH-
)1-2--((CR3R4)q1--)--,
--((CR3R4)q1)1--(NHCO--(CR3R4)q1--CONH-
)1-2--((CR3R4)q1)1 or
--((CR3R4)q1)1--(CONH--(CR3R4)q1--NHCO-
)1-2--((CR3R4)q1)1 where R3 and R4
independently of each other can be H, --COOH, and R3 and R4 can
be different at each carbon, and q1 is 1-6-; and a bond;with the
proviso that Q1-Q4 are different;X1, X2 and X3
are independently selected from: O; a bond; ##STR00072## where R is
hydrogen or --(CH2)p--COOH, --(CH2)p--SO3H,
--(CH2)p--PO3H2, --(CH2)p--O--SO3H;
--(CH2)p--O--PO3H2; or
--(CH2)p-tetrazol-5-yl, where each p independently of the other
p's is an integer in the range of 1 to 6; andZ is selected from: --COOH;
--CO-Asp; --CO-Glu; --CO-Gly; --CO-Sar; --CH(COOH)2;
--N(CH2COOH)2; --SO3H; --OSO3H; --OPO3H2,
--PO3H2 and -tetrazol-5-yland any Zn2+ complex thereof.

11. The insulin derivative according to claim 10, wherein s is in the
range of 2-12, 2-4 or 2-3

12. The insulin derivative according to claim 10, wherein s is preferably
1.

13. The insulin derivative according to claim 10, wherein Z is --COOH.

14. The insulin derivative according to claim 1, wherein the parent
insulin is a desB30 human insulin analogue.

17. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a therapeutically
effective amount of an insulin derivative, said insulin derivative having
a side chain attached either to the α-amino group of the N-terminal
amino acid residue of B chain or to an s-amino group of a Lys residue
present in the B chain of the parent insulin molecule via an amide bond
which side chain comprises one or more residues of ethylenglycol,
propyleneglycol and/or butyleneglycol containing independently at each
termini a group selected from --NH2 and --COOH; a fatty diacid
moiety with from 4 to 22 carbon atoms, at least one free carboxylic acid
group or a group which is negatively charged at neutral pH; and possible
linkers which link the individual components in the side chain together
via amide or ether bonds, said linkers optionally comprising a free
carboxylic acid group together with a pharmaceutically acceptable
carrier.

18. A method of treating diabetes in a patient in need of such a
treatment, comprising administering to the patient a therapeutically
effective amount of an insulin derivative, said insulin derivative having
a side chain attached either to the α-amino group of the N-terminal
amino acid residue of B chain or to an s-amino group of a Lys residue
present in the B chain of the parent insulin molecule via an amide bond
which side chain comprises one or more residues of ethylenglycol,
propyleneglycol and/or butyleneglycol containing independently at each
termini a group selected from --NH2 and --COOH; a fatty diacid
moiety with from 4 to 22 carbon atoms, at least one free carboxylic acid
group or a group which is negatively charged at neutral pH; and possible
linkers which link the individual components in the side chain together
via amide or ether bonds, said linkers optionally comprising a free
carboxylic acid group in mixture with an insulin or an insulin analogue
which has a rapid onset of action, together with a pharmaceutically
acceptable carrier.

[0002]The present invention relates to novel human insulin derivatives
which are soluble at physiological pH values and have a prolonged profile
of action. The invention also relates to methods of providing such
derivatives, to pharmaceutical compositions containing them, to a method
of treating diabetes and hyperglycaemia using the insulin derivatives of
the invention and to the use of such insulin derivatives in the treatment
of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

[0003]Currently, the treatment of diabetes, both type 1 diabetes and type
2 diabetes, relies to an increasing extent on the so-called intensive
insulin treatment. According to this regimen, the patients are treated
with multiple daily insulin injections comprising one or two daily
injections of long acting insulin to cover the basal insulin requirement
supplemented by bolus injections of a rapid acting insulin to cover the
insulin requirement related to meals.

[0004]Long acting insulin compositions are well known in the art. Thus,
one main type of long acting insulin compositions comprises injectable
aqueous suspensions of insulin crystals or amorphous insulin. In these
compositions, the insulin compounds utilized typically are protamine
insulin, zinc insulin or protamine zinc insulin.

[0005]Certain drawbacks are associated with the use of insulin
suspensions. Thus, in order to secure an accurate dosing, the insulin
particles must be suspended homogeneously by gentle shaking before a
defined volume of the suspension is withdrawn from a vial or expelled
from a cartridge. Also, for the storage of insulin suspensions, the
temperature must be kept within more narrow limits than for insulin
solutions in order to avoid lump formation or coagulation.

[0006]Another type of long acting insulin compositions are solutions
having a pH value below physiological pH from which the insulin will
precipitate because of the rise in the pH value when the solution is
injected. A drawback with these solutions is that the particle size
distribution of the precipitate formed in the tissue on injection, and
thus the release profile of the medication, depends on the blood flow at
the injection site and other parameters in a somewhat unpredictable
manner. A further drawback is that the solid particles of the insulin may
act as a local irritant causing inflammation of the tissue at the site of
injection.

[0007]Human insulin has three primary amino groups: the N-terminal group
of the A-chain and of the B-chain and the ε-amino group of
LysB29. Several insulin derivatives which are substituted in one or more
of these groups are known in the prior art. Thus, U.S. Pat. No. 3,528,960
(Eli Lilly) relates to N-carboxyaroyl insulins in which one, two or three
primary amino groups of the insulin molecule has a carboxyaroyl group.

[0008]According to GB Patent No. 1,492,997 (Nat. Res. Dev. Corp.), it has
been found that insulin with a carbamyl substitution at
N.sup.εB29 has an improved profile of hypoglycaemic effect.

[0009]JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.)
discloses insulin wherein a fatty acid is bound to the amino group of
PheB1 or to the ε-amino group of LysB29 or to both of these. The
stated purpose of the derivatisation is to obtain a pharmacologically
acceptable, stable insulin preparation.

[0010]Insulins, which in the B30 position have an amino acid having at
least five carbon atoms which cannot necessarily be coded for by a
triplet of nucleotides, are described in JP laid-open patent application
No. 57-067548 (Shionogi). The insulin analogues are claimed to be useful
in the treatment of diabetes mellitus, particularly in patients who are
insulin resistant due to generation of bovine or porcine insulin
antibodies.

[0011]WO 95/07931 (Novo Nordisk A/S) discloses human insulin derivatives
wherein the ε-amino group of LysB29 has a lipophilic substituent.
These insulin derivatives have a prolonged profile of action and are
soluble at physiological pH values.

[0012]EP 894095 discloses insulin derivatives wherein the N-terminal group
of the B-chain and/or the ε-amino group of Lys in position B28,
B29 or B30 has a substituent of the formula --CO--W--COOH where W can be
a long chain hydrocarbon group. These insulin derivatives have a
prolonged profile of action and are soluble at physiological pH values.

[0013]Unfortunately, many diabetics are unwilling to undertake intensive
therapy due to the discomfort associated with the many injections
required to maintain close control of glucose levels. This type of
therapy can be both psychologically and physically painful. Upon oral
administration, insulin is rapidly degraded in the gastro intestinal
tract and is not absorbed into the blood stream. Therefore, many
investigators have studied alternate routes for administering insulin,
such as oral, rectal, transdermal, and nasal routes. Thus far, however,
these routes of administration have not resulted in effective insulin
absorption.

[0014]Efficient pulmonary delivery of a protein is dependent on the
ability to deliver the protein to the deep lung alveolar epithelium.
Proteins that are deposited in the upper airway epithelium are not
absorbed to a significant extent. This is due to the overlying mucus
which is approximately 30-40 μm thick and acts as a barrier to
absorption. In addition, proteins deposited on this epithelium are
cleared by mucociliary transport up the airways and then eliminated via
the gastrointestinal tract. This mechanism also contributes substantially
to the low absorption of some protein particles. The extent to which
proteins are not absorbed and instead eliminated by these routes depends
on their solubility, their size, as well as other less understood
characteristics.

[0015]It is however well recognised that the properties of peptides can be
enhanced by grafting organic chain-like molecules onto them. Such
grafting can improve pharmaceutical properties such as half life in
serum, stability against proteolytical degradation, and reduced
immunogenicity.

[0016]The organic chain-like molecules often used to enhance properties
are polyethylene glycol-based or polyethylene based chains, i.e., chains
that are based on the repeating unit --CH2CH2O--. Hereinafter,
the abbreviation "PEG" is used for polyethyleneglycol.

[0017]Classical PEG technology takes advantage of providing polypeptides
with increased size (Stoke radius) by attaching a soluble organic
molecule to the polypeptide (Kochendoerfer, G., et al., Science (299)
884-, 2003). This technology leads to reduced clearance in man and
animals of a hormone polypeptide compared to the native polypeptide.
However this technique is often hampered by reduced potency of the
hormone polypeptides subjected to this technique (Hinds, K., et al.,
Bioconjugate Chem. (11), 195-, 2000). WO 02/20033 discloses a general
method for the synthesis of well defined polymer modified peptides.

[0018]However, there is still a need for insulins having a more prolonged
profile of action than the insulin derivatives known up till now and
which at the same time are soluble at physiological pH values and have a
potency which is comparable to that of human insulin. Furthermore, there
is need for further insulin formulations which are well suited for
pulmonary application.

SUMMARY OF THE INVENTION

[0019]The present invention is based on the recognition that acylation of
insulin with one or more residues of ethylenglycol, propyleneglycol
and/or butyleneglycol in combination with fatty diacid residues has
surprisingly shown a good bioavailability.

[0020]Organic chain-like molecules, which can be used to enhance
properties, are poly-ethyleneglycol based, polypropyleneglycol based or
polybutyleneglycol based chains, i.e., chains that are based on the
repeating unit CH2CH2O--, CH2CH2CH2O-- or
CH2CH2CH2CH2O--. Hereinafter, the abbreviation "PEG"
is used for polyethyleneglycol, "PPG" is used for polypropyleneglycol and
"PBG" is used for polybutyleneglycol.

[0021]In one aspect the present invention is related to insulin
derivatives having a side chain attached either to the α-amino
group of the N-terminal amino acid residue of the B chain or to an
ε-amino group of a Lys residue present in the B chain of the
parent insulin molecule via an amide bond which side chain comprises one
or more residues of ethylenglycol, propyleneglycol and/or butyleneglycol
containing independently at each termini a group selected from --NH2
and --COOH; a fatty diacid moiety with 4 to 22 carbon atoms; at least one
free carboxylic acid group or a group which is negatively charged at
neutral pH; and possible linkers which link the individual components in
the side chain together via amide, ether or amine bonds, said linkers
optionally comprising a free carboxylic acid group.

[0022]In one aspect the insulin derivatives contain a difunctional PEG,
PPG or PBG group that has from 2 to 20; from 2 to 10 or from 2 to 5
residues of ethyleneglycol, propyleneglycol or butyleneglycol,
respectively.

[0023]In one aspect the side chain of the insulin derivative comprise one
single residue of ethyleneglycol.

[0024]In one aspect the side chain of the insulin derivative comprise one
single residue of propyleneglycol.

[0025]In one aspect the side chain of the insulin derivative comprise one
single residue of butyleneglycol.

[0026]In one aspect the side chain of the insulin derivative has single
residues of ethylenglycol, propyleneglycol or butyleneglycol alone or in
combination.

[0027]In one aspect the side chain of the insulin derivative has one
residue of propyleneglycol and one residue of butyleneglycol.

[0028]In one aspect the fatty diacid comprises from 4 to 22 carbon atoms
in the carbon chain.

[0029]In one aspect the fatty diacid comprises from 6 to 22, from 8 to 20,
from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16, from 8 to 22,
from 8 to 17 or from 8 to 15 carbon atoms in the carbon chain.

[0030]In one aspect the linker is an amino acid residue, a peptide chain
of 2-4 amino acid residues or has the motif is α-Asp; β-Asp;
α-Glu; δ-Glu; α-hGlu; δ-hGlu;
--N(CH2COOH)CH2CO--;
--N(CH2CH2COOH)CH2CH2CO--;
--N(CH2COOH)CH2CH2CO-- or
--N(CH2CH2COOH)CH2CO--.

[0031]In one aspect the Lys residue in the B chain will be position B3,
B29 or in one of positions B23-B30.

[0032]In another aspect the invention is related to an insulin derivative
having the formula

##STR00001##

wherein Ins is the parent insulin moiety which via the α-amino group
of the N-terminal amino acid residue of the B chain or an ε-amino
group of a Lys residue present in the B chain of the insulin moiety is
bound to the CO-- group in the side chain via an amide bond; each n is
independently 0, 1, 2, 3, 4, 5 or 6; [0033]Q1, Q2, Q3, and
Q4 independently of each other can be
[0034](CH2CH2O)s--; (CH2CH2CH2O)s--;
(CH2CH2CH2CH2O)s--;
(CH2CH2OCH2CH2CH2CH2O)s-- or
(CH2CH2CH2OCH2CH2CH2CH2O)s--
where s is 1-20 [0035]--(CH2)r-- where r is an integer from 4
to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 --CH═CH--
groups and a number of --CH2-- groups sufficient to give a total
number of carbon atoms in the chain in the range of 4 to 22;
[0036]--(CH2)t-- or --(CH2OCH2)t--, where t is
an integer from 1 to 6; [0037]--(CR1R2)q--, where R1
and R2 independently of each other can be H, --COOH,
(CH2)1-6COOH and R1 and R2 can be different at each
carbon, and q is 1-6,
[0038]--((CR3R4)q1)1--(NHCO--(CR3R4)q1-
--NHCO)1-2--((CR3R4)q1)1 or
--((CR3R4)q1)1--(CONH--(CR3R4)q1--CONH-
)1-2--((CR3R4)q1--)--,
--((CR3R4)q1)1--(NHCO--(CR3R4)q1--CONH-
)1-2--((CR3R4)q1)1 or
--((CR3R4)q1)1--(CONH--(CR3R4)q1--NHCO-
)1-2--((CR3R4)q1)1 where R3 and R4
independently of each other can be H, --COOH, and R3 and R4 can
be different at each carbon, and q1 is 1-6-, or [0039]a bond;

[0040]with the proviso that Q1-Q4 are different;

[0041]X1, X2 and X3 are independently [0042]O; [0043]a
bond; or

##STR00002##

[0044]where R is hydrogen or --(CH2)p--COOH,
--(CH2)p--SO3H, --(CH2)p--PO3H2,
--(CH2)p--O--SO3H; --(CH2)p--O--PO3H2;
or --(CH2)p-tetrazol-5-yl, where each p independently of the
other p's is an integer in the range of 1 to 6; and

[0045]Z is:

[0046]--COOH;

[0047]--CO-Asp;

[0048]--CO-Glu;

[0049]--CO-Gly;

[0050]--CO-Sar;

[0051]--CH(COOH)2,

[0052]--N(CH2COOH)2;

[0053]--SO3H

[0054]--OSO3H

[0055]--OPO3H2

[0056]--PO3H2 or

[0057]-tetrazol-5-yl

[0058]and any Zn2+ complex thereof.

[0059]Where mentioned that R1, R2, R3 and R4 can be
different at each carbon is meant that R1, R2, R3 and
R4 can be different for each value of q or q1.

[0060]In one aspect r is from 6 to 22, from 8 to 20, from 8 to 18, from 4
to 18, from 6 to 18 from 8 to 16 from 8 to 22 from 8 to 17 from 8 to 15.

[0061]In another aspect s is in the range of 2-12, 2-4 or 2-3.

[0062]In another aspect s is 1.

[0063]In one aspect n is from 1-6, from 2-6, from 2-5, from 2-4, from 0-2
or from 2-3.

[0064]In one aspect q is from 1-5, from1-4, from 1-3 or from 1-2.

[0065]In one aspect q1 is from 1-5, from 1-4, from 1-3 or from 1-2.

[0066]In one aspect t is from 1-6, from 1-5, from 1-4, from 1-3 or from
1-2.

[0067]In one aspect Z is --COOH.

[0068]In one aspect Z is --CO-Asp.

[0069]In another aspect Z is --CO-Glu.

[0070]In another aspect Z is --CO-Gly.

[0071]In another aspect Z is --CO-Sar.

[0072]In another aspect Z is --CH(COOH)2.

[0073]In another aspect Z is --N(CH2COOH)2.

[0074]In another aspect Z is --SO3H.

[0075]In another aspect Z is --PO3H.

[0076]In another aspect Z is O--SO3H;

[0077]In another aspect Z is O--PO3H2;

[0078]In another aspect Z is tetrazol-5-yl.

[0079]In a further aspect the parent insulin is a desB30 human insulin
analogue.

[0082]Insulin derivatives according to the invention may be provided in
the form of essentially zinc free compounds or in the form of zinc
complexes. When zinc complexes of an insulin derivative according to the
invention are provided, two Zn2+ ions, three Zn2+ ions or four
Zn2+ ions can be bound to each insulin hexamer. Solutions of zinc
complexes of the insulin derivatives will contain mixtures of such
species.

[0083]In a further aspect the invention is related to a pharmaceutical
composition comprising a therapeutically effective amount of an insulin
derivative according to the invention together with a pharmaceutically
acceptable carrier can be provided for the treatment of type 1 diabetes,
type 2 diabetes and other states that cause hyperglycaemia in patients in
need of such a treatment. An insulin derivative according to the
invention can be used for the manufacture of a pharmaceutical composition
for use in the treatment of type 1 diabetes, type 2 diabetes and other
states that cause hyperglycaemia.

[0084]In a further aspect of the invention, there is provided a
pharmaceutical composition for treating type 1 diabetes, type 2 diabetes
and other states that cause hyperglycaemia in a patient in need of such a
treatment, comprising a therapeutically effective amount of an insulin
derivative according to the invention in mixture with an insulin or an
insulin analogue which has a rapid onset of action, together with
pharmaceutically acceptable carriers and additives.

[0085]In a further aspect the invention is related to a pulmonary
application for treating type 1 diabetes, type 2 diabetes and other
states that cause hyperglycaemia in a patient in need of such a
treatment, comprising a therapeutically effective amount of an insulin
derivative according to the invention optionally in mixture with an
insulin or an insulin analogue which has a rapid onset of action,
together with pharmaceutically acceptable carriers and additives.

[0086]In one aspect the invention provides a pharmaceutical composition
being a mixture of an insulin derivative according to the invention and a
rapid acting insulin analogue selected group consisting of AspB28 human
insulin; LysB28 ProB29 human insulin and LysB3 GluB29 human insulin.

[0087]The insulin derivative according to the invention and the rapid
acting insulin analogue can be mixed in a ratio from about 90/10%; about
70/30% or about 50/50%.

[0088]In a further aspect of the invention, there is provided a method of
treating type 1 diabetes, type 2 diabetes and other states that cause
hyperglycaemia in a patient in need of such a treatment, comprising
administering to the patient a therapeutically effective amount of an
insulin derivative according to the invention together with a
pharmaceutically acceptable carrier and pharmaceutical acceptable
additives.

[0089]In a further aspect of the invention, there is provided a method of
treating type 1 diabetes, type 2 diabetes and other states that cause
hyperglycaemia in a patient in need of such a treatment, comprising
administering to the patient a therapeutically effective amount of an
insulin derivative according to the invention in mixture with an insulin
or an insulin analogue which has a rapid onset of action, together with a
pharmaceutically acceptable carrier and pharmaceutical acceptable
additives.

[0090]In another aspect of the invention the insulin derivatives has a
side chain attached either to the α-amino group of the N-terminal
amino acid residue of B chain or to an ε-amino group of a Lys
residue present in the B chain of the parent insulin molecule via an
amide bond which side chain comprises a monodisperse, diffunctionel PEG
group containing independently at each termini a group selected from
--OH; --NH2 and --COOH; a fatty diacid moiety with 4 to 22 carbon
atoms, at least one free carboxylic acid group or a group which is
negatively charged at neutral pH; and possible linkers which link the
individual components in the side chain together via amide, ether or
amine bonds, said linkers optionally comprising a free carboxylic acid
group.

[0091]In another aspect of the invention the PEG group of the insulin
derivative has from 1 to 20; from 1 to 10 or from 1 to 5 ethylene
residues.

[0092]In another aspect of the invention the insulin derivatives has a
side chain attached either to the α-amino group of the N-terminal
amino acid residue of B chain or to an ε-amino group of a Lys
residue present in the B chain of the parent insulin molecule via an
amide bond which side chain comprises a monodisperse, diffunctionel PEG
group containing independently at each termini a group selected from
--OH; --NH2 and --COOH; a fatty diacid moiety with 4 to 22 carbon
atoms, at least one free carboxylic acid group or a group which is
negatively charged at neutral pH; and possible linkers which link the
individual components in the side chain together via amide, ether or
amine bonds, said linkers optionally comprising a free carboxylic acid
group.

[0093]In a further aspect of the invention the insulin derivatives
comprises a difunctionel PEG group which has from 1 to 20; from 1 to 10
or from 1 to 5 ethylene units.

[0094]In a further aspect of the invention the insulin derivatives
comprises a fatty diacid which comprises from 4 to 22 carbon atoms in the
carbon chain.

[0095]In a further aspect of the invention the insulin derivatives
comprises a fatty acid, wherein the fatty diacid comprises from 6 to 22,
from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16,
from 8 to 22, from 8 to 17 or from 8 to 15 carbon atoms in the carbon
chain.

[0096]In a further aspect of the invention the insulin derivatives
comprises a linker wherein the linker is an amino acid residue, a peptide
chain of 2-4 amino acid residues or has the motif α-Asp,
β-Asp, α-Glu, γ-Glu, α-hGlu, δ-hGlu,
--N(CH2COOH)CH2CO--,
--N(CH2CH2COOH)CH2CH2CO--,
--N(CH2COOH)CH2CH2CO-- or
--N(CH2CH2COOH)CH2CO--

[0097]In a further aspect of the invention the insulin derivatives
comprises a Lys residue wherein the Lys residue in the B chain of the
parent insulin in in either position B3 or in one of positions B23-30.

[0098]In a further aspect of the invention the insulin derivatives has the
formula

##STR00003##

wherein lns is the parent insulin moiety which via the α-amino group
of the N-terminal amino acid residue of the B chain or an ε-amino
group of a Lys residue present in the B chain of the insulin moiety is
bound to the CO-- group in the side chain via an amide bond; [0099]each n
is independently 0, 1, 2, 3, 4, 5 or 6; [0100]Q1, Q2, Q3,
and Q4 independently of each other can be
[0101](CH2CH2O)s-- where s is 1-20,
[0102]--(CH2)r-- where r is an integer from 4 to 22; or a
divalent hydrocarbon chain comprising 1, 2 or 3 --CH═CH-- groups and
a number of --CH2-- groups sufficient to give a total number of
carbon atoms in the chain in the range of 4 to 22;
[0103]--(CH2)t-- or --(CH2OCH2)t--, where t is
an integer from 1 to 6; [0104]--(CR1R2)q--, where R1
and R2 independently of each other can be H, --COOH, and R1 and
R2 can be different at each carbon, and q is 1-6,
[0105]--((CR3R4)q1)1--(NHCO--(CR3R4)q1-
--NHCO)1-2--((CR3R4)q1)1 or
--((CR3R4)q1)1--(CONH--(CR3R4)q1--CONH-
)1-2--((CR3R4)q1--, where R3 and R4
independently of each other can be H, --COOH, and R3 and R4 can
be different at each carbon, and q1 is 1-6-, or [0106]a bond;

[0107]with the proviso that Q1-Q4 are different;

[0108]X and V and G are independently [0109]O; [0110]a bond; or

##STR00004##

[0111]where R is hydrogen or --(CH2)p--COOH,
--(CH2)p--SO3H, --(CH2)p--PO3H2,
--(CH2)p--O--SO3H; --(CH2)p--O--PO3H2;
or --(CH2)p-tetrazolyl, where each p independently of the other
p's is an integer in the range of 1 to 6; and

[0112]Z is:

[0113]--COOH;

[0114]--CO-Asp;

[0115]--CO-Glu;

[0116]--CO-Gly;

[0117]--CO-Sar;

[0118]--CH(COOH)2,

[0119]--N(CH2COOH)2;

[0120]--SO3H

[0121]--OSO3H

[0122]--OPO3H2

[0123]--PO3H2 or

[0124]-tetrazolyl.

[0125]In a further aspect of the invention the insulin derivatives
according to the formula, s is from 6 to 22, from 8 to 20, from 8 to 18,
from 4 to 18, from 6 to 18, from 8 to 16, from 8 to 22, from 8 to 17 or
from 8 to 15.

[0126]In a further aspect of the invention the insulin derivatives
according to the formula s is from 1-20, from 1-10 or from 1-5.

[0127]In a further aspect of the invention the insulin derivative
according to the formula, Z is --COOH.

[0128]In a further aspect of the invention the insulin derivative
according to the invention, the parent insulin is a desB30 human insulin
analogue.

[0131]In a further aspect of the invention there is provided a
pharmaceutical composition for the treatment of diabetes in a patient in
need of such treatment, comprising a therapeutically effective amount of
an insulin derivative according to the invention together with a
pharmaceutically acceptable carrier.

[0132]In a further aspect of the invention there is provided a
pharmaceutical composition for the treatment of diabetes in a patient in
need of such treatment, comprising a therapeutically effective amount of
an insulin derivative according to the invention in mixture with an
insulin or an insulin analogue which has a rapid onset of action,
together with a pharmaceutically acceptable carrier.

[0133]In a further aspect of the invention there is provided a
pharmaceutical composition according to the invention intended for
pulmonal administration.

[0134]In a further aspect of the invention there is provided a method of
treating diabetes in a patient in need of such a treatment, comprising
administering to the patient a therapeutically effective amount of an
insulin derivative according to claim 1 together with a pharmaceutically
acceptable carrier.

[0135]In a further aspect of the invention there is provided a method of
treating diabetes in a patient in need of such a treatment, comprising
administering to the patient a therapeutically effective amount of an
insulin derivative according to claim 1 in mixture with an insulin or an
insulin analogue which has a rapid onset of action, together with a
pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

[0136]The present insulin derivatives are characterized by having a side
chain attached to a Lys group in the B chain or to the N-terminal amino
group in the B-chain of the parent insulin molecule which side chain
comprises one or more residues of ethylenglycol, propylene-glycol and/or
butyleneglycol and a fatty diacid moiety.

[0137]The insulin derivative according to the invention is furthermore
characterized in having at least one free carboxylic acid group in the
side chain and may comprise up to 2, 3 or 4 free carboxylic acid groups
or a group which is negatively charged at neutral pH.

[0138]The insulin derivatives will only contain one lysine residue. This
lysine residue may either be in position B29 as in human insulin or in
one of position B3, B30 or B23 to B28.

[0139]The residues of ethylenglycol, propyleneglycol and/or butyleneglycol
will have any combination of the three groups --OH; --NH2 and --COOH
at each end. The residues of ethylenglycol, propyleneglycol and/or
butyleneglycol will typically be in the form of an ethyleneglycol residue
followed by a butyleneglycol residue or have a chain length of 2 to 20
PEG, PPG or PBG residues corresponding to a molecular weight of about 200
to 800.

[0140]The residues of ethylenglycol, propyleneglycol and/or butyleneglycol
will typically be in the form of an ethyleneglycol residue followed by a
butylen residue
--(CH2CH2OCH2CH2CH2CH2O)m where m is 1
to 20.

[0141]The difunctional PEG or PPG or PBG will have any combination of the
three groups --OH; --NH2, and --COOH at each end and will typically
have a chain length of 1 to 20 PEG residues corresponding to a molecular
weight of about 200 to 1000.

[0142]Non limiting examples of amino PEG moieties are
H2N--(CH2)u--(OCH2CH2)m--O(CH2)u--
-COOH and H2N--(CH2)v--NH--CO--(CH2)u--(OCH2-
CH2)m--(CH2)u--COOH, where u are independently 1 to 6,
m is 2 to 20 and v is 1 to 6.

[0143]Non limiting examples of amino PPG moieties are
H2N--(CH2)u--(OCH2CH2CH2)m--O(CH2-
)u--COOH and
H2N--(CH2)v--NH--CO--(CH2)u--(OCH2CH2C-
H2)m--(CH2)u--COOH, where u are independently 1 to 6,
m is 2 to 20 and v is 1 to 6.

[0144]Non limiting examples of amino PBG moieties are
H2N--(CH2)u--(OCH2CH2CH2CH2)m--O(-
CH2)u--COOH and
H2N--(CH2)v--NH--CO--(CH2)u--(OCH2CH2C-
H2CH2)m--(CH2)u--COOH, where u are independently
1 to 6, m is 2 to 20 and v is 1 to 6.

[0145]The fatty diacid will typically comprise from 4 to 22, from 6 to 22,
from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16,
from 8 to 22, from 8 to 12, from 8 to 10, from 8 to 17 or from 8 to 15
carbon atoms in the carbon chain.

[0147]The insulin moiety--in the present text also referred to as the
parent insulin--or insulin derivative according to the invention can be a
naturally occurring insulin such as human insulin or porcine insulin.
Alternatively, the parent insulin can be an insulin analogue.

[0148]In one group of parent insulin analogues, the amino acid residue at
position A21 is Asn.

[0149]In another group of parent insulin analogues, the amino acid residue
at position B1 has been deleted. A specific example from this group of
parent insulin analogues is desB1 human insulin.

[0150]In another group of parent insulin analogues, the amino acid residue
at position B30 has been deleted. A specific example from this group of
parent insulin analogues is desB30 human insulin.

[0151]In another group of parent insulin analogues, the amino acid residue
at position B28 is Asp. A specific example from this group of parent
insulin analogues is AspB28 human insulin.

[0152]In another group of parent insulin analogues, the amino acid residue
at position B28 is Lys and the amino acid residue at position B29 is Pro.
A specific example from this group of parent insulin analogues is LysB28
ProB29 human insulin.

[0153]In another group of parent insulin analogues the amino acid residue
in position B30 is Lys and the amino acid residue in position B29 is any
codable amino acid except Cys, Arg and Lys. An example is an insulin
analogue where the amino acid residue at position B29 is Thr and the
amino acid residue at position B30 is Lys. A specific example from this
group of parent insulin analogues is ThrB29 LysB30 human insulin.

[0154]In another group of parent insulin analogues, the amino acid residue
at position B3 is Lys and the amino acid residue at position B29 is Glu.
A specific example from this group of parent insulin analogues is LysB3
GluB29 human insulin.

[0155]The linkers will typically be an amino acid residue or a chain of
amino acid residue comprising up to four amino acids. Thus, the linker
may be selected from the group consisting of α-Asp; β-Asp;
α-Glu; γ-Glu; α-hGlu; δ-hGlu;
--N(CH2COOH)CH2CO--,
--N(CH2CH2COOH)CH2CH2CO--;
--N(CH2COOH)CH2CH2CO-- or
--N(CH2CH2COOH)CH2CO--

[0156]In a further aspect the linker can be a chain composed of two amino
acid residues of which one has from 4 to 10 carbon atoms and a carboxylic
acid group in the side chain while the other has from 2 to 11 carbon
atoms but no free carboxylic acid group. The amino acid residue with no
free carboxylic acid group can be a neutral, codable α-amino acid
residue. Examples of such linkers are are: α-Asp-Gly;
Gly-α-Asp; β-Asp-Gly; Gly-β-Asp; α-Glu-Gly;
Gly-α-Glu; γ-Glu-Gly; Gly-γ-Glu; α-hGlu-Gly;
Gly-α-hGlu; δ-hGlu-Gly; and Gly-δ-hGlu.

[0158]In a further aspect the linker is a chain composed of three amino
acid residues, independently having from 4 to 10 carbon atoms, the amino
acid residues of the chain being selected from the group of residues
having a neutral side chain and residues having a carboxylic acid group
in the side chain so that the chain has at least one residue which has a
carboxylic acid group in the side chain. In one aspect, the amino acid
residues are codable residues.

[0159]In a further aspect, the linker is a chain composed of four amino
acid residues, independently having from 4 to 10 carbon atoms, the amino
acid residues of the chain being selected from the group having a neutral
side chain and residues having a carboxylic acid group in the side chain
so that the chain has at least one residue which has a carboxylic acid
group in the side chain. In one aspect, the amino acid residues are
codable residues.

[0160]Examples of insulin derivatives according to the invention are the
following compounds:

[0378]In a further aspect, the present invention relates to insulin
derivatives which have an overall hydrophobicity which is essentially
similar to that of human insulin.

[0379]In a further aspect, the insulin derivatives of the present
invention have a hydrophobic index, k'rel, which is in the range from
about 0.02 to about 10, from about 0.1 to about 5; from about 0.5 to
about 5; from about 0.2 to about 2; from about 0.2 to about 1; from about
0.1 to about 2; or from about 0.5 to about 2.

[0380]According to one aspect of the present invention, the insulin
derivatives will comprise a side chain of general formula (I) as defined
above which have at least one free carboxylic acid group and according to
a further aspect, the side chain will optionally hold one or more free
carboxylic acid groups.

[0381]The hydrophobicity (hydrophobic index) of the insulin derivatives of
the invention relative to human insulin, k'rel, was measured on a
LiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic
elution at 40° C. using mixtures of A) 0.1 M sodium phosphate
buffer, pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in
water as eluents. The elution was monitored by following the UV
absorption of the eluate at 214 nm. Void time, t0, was found by
injecting 0.1 mM sodium nitrate. Retention time for human insulin,
thuman, was adjusted to at least 2t0 by varying the ratio
between the A and B solutions.
k'rel=(t.sub.derivative-t0)/(thuman-t0).

[0382]In another aspect, the invention relates to a pharmaceutical
composition comprising an insulin derivative according to the invention
which is soluble at physiological pH values.

[0383]In another aspect, the invention relates to a pharmaceutical
composition comprising an insulin derivative according to the invention
which is soluble at pH values in the interval from about 6.5 to about
8.5.

[0384]In another aspect, the invention relates to a pharmaceutical
composition with a prolonged profile of action which comprises an insulin
derivative according to the invention.

[0385]In another aspect, the invention relates to a pharmaceutical
composition which is a solution containing from about 120 nmol/ml to
about 2400 nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from
about 400 nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about
2400 nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an
insulin derivative according to the invention or of a mixture of the
insulin derivative according to the invention with a rapid acting insulin
analogue.

[0386]The starting product for the acylation, the parent insulin or
insulin analogue or a precursor thereof can be produced by either
well-know peptide synthesis or by well known recombinant production in
suitable transformed microorganisms. Thus the insulin starting product
can be produced by a method which comprises culturing a host cell
containing a DNA sequence encoding the polypeptide and capable of
expressing the polypeptide in a suitable nutrient medium under conditions
permitting the expression of the peptide, after which the resulting
peptide is recovered from the culture.

[0387]As an example desB30 human insulin can be produced from a human
insulin precursor B(1-29)-Ala-Ala-Lys-A(1-21) which is produced in yeast
as disclosed in U.S. Pat. No. 4,916,212. This insulin precursor can then
be converted into desB30 human insulin by ALP cleavage of the Ala-Ala-Lys
peptide chain to give desB30 human insulin which can then be acylated to
give the present insulintives.

[0388]The medium used to culture the cells may be any conventional medium
suitable for growing the host cells, such as minimal or complex media
containing appropriate supplements. Suitable media are available from
commercial suppliers or may be prepared according to published recipes
(e.g. in catalogues of the American Type Culture Collection). The peptide
produced by the cells may then be recovered from the culture medium by
conventional procedures including separating the host cells from the
medium by centrifugation or filtration, precipitating the proteinaceous
components of the supernatant or filtrate by means of a salt, e.g.
ammonium sulphate, purification by a variety of chromatographic
procedures, e.g. ion exchange chromatography, gel filtration
chromatography, affinity chromatography, or the like, dependent on the
type of peptide in question.

[0389]The DNA sequence encoding the parent insulin may suitably be of
genomic or cDNA origin, for instance obtained by preparing a genomic or
cDNA library and screening for DNA sequences coding for all or part of
the polypeptide by hybridisation using synthetic oligonucleotide probes
in accordance with standard techniques (see, for example, Sambrook, J,
Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence
encoding the parent insulin may also be prepared synthetically by
established standard methods, e.g. the phosphoamidite method described by
Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the
method described by Matthes et al., EMBO Journal 3 (1984), 801-805. The
DNA sequence may also be prepared by polymerase chain reaction using
specific primers, for instance as described in U.S. Pat. No. 4,683,202 or
Saiki et al., Science 239 (1988), 487-491.

[0390]The DNA sequence may be inserted into any vector which may
conveniently be subjected to recombinant DNA procedures, and the choice
of vector will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating vector,
i.e. a vector which exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g. a plasmid.
Alternatively, the vector may be one which, when introduced into a host
cell, is integrated into the host cell genome and replicated together
with the chromosome(s) into which it has been integrated.

[0391]The vector is preferably an expression vector in which the DNA
sequence encoding the parent insulin is operably linked to additional
segments required for transcription of the DNA, such as a promoter. The
promoter may be any DNA sequence which shows transcriptional activity in
the host cell of choice and may be derived from genes encoding proteins
either homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA encoding the parent
insulin in a variety of host cells are well known in the art, cf. for
instance Sambrook et al., supra.

[0392]The DNA sequence encoding the parent insulin may also, if necessary,
be operably connected to a suitable terminator, polyadenylation signals,
transcriptional enhancer sequences, and translational enhancer sequences.
The recombinant vector of the invention may further comprise a DNA
sequence enabling the vector to replicate in the host cell in question.

[0393]The vector may also comprise a selectable marker, e.g. a gene the
product of which complements a defect in the host cell or one which
confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,
chloramphenicol, neomycin, hygromycin or methotrexate.

[0394]To direct a peptide of the present invention into the secretory
pathway of the host cells, a secretory signal sequence (also known as a
leader sequence, prepro sequence or pre sequence) may be provided in the
recombinant vector. The secretory signal sequence is joined to the DNA
sequence encoding the peptide in the correct reading frame. Secretory
signal sequences are commonly positioned 5' to the DNA sequence encoding
the peptide. The secretory signal sequence may be that normally
associated with the peptide or may be from a gene encoding another
secreted protein.

[0395]The procedures used to ligate the DNA sequences coding for the
parent insulin, the promoter and optionally the terminator and/or
secretory signal sequence, respectively, and to insert them into suitable
vectors containing the information necessary for replication, are well
known to persons skilled in the art (cf., for instance, Sambrook et al.,
supra).

[0396]The host cell into which the DNA sequence or the recombinant vector
is introduced may be any cell which is capable of producing the present
peptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
Examples of suitable host cells well known and used in the art are,
without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK
or CHO cell lines.

[0397]The parent insulin molecule is then converted into the insulin
derivatives of the invention by introducing of the relevant side chain in
either the B1 position or in the chosen Lys position in the B-chain. The
side chain can be introduced by any convenient method and many methods
are disclosed in the prior art for acylation of an amino group. More
details will appear from the following examples.

Pharmaceutical Compositions

[0398]The insulin derivatives of this invention of the claimed formula
can, for example, be administered subcutaneously, orally, or pulmonary.

[0399]For subcutaneous administration, the compounds of the formula are
formulated analogously with the formulation of known insulins.
Furthermore, for subcutaneous administration, the compounds of the
formula are administered analogously with the administration of known
insulins and, generally, the physicians are familiar with this procedure.

[0400]The insulin derivatives of this invention may be administered by
inhalation in a dose effective manner to increase circulating insulin
levels and/or to lower circulating glucose levels. Such administration
can be effective for treating disorders such as diabetes or
hyperglycemia. Achieving effective doses of insulin requires
administration of an inhaled dose of insulin derivative of this invention
of more than about 0.5 μg/kg to about 50 μg/kg. A therapeutically
effective amount can be determined by a knowledgeable practitioner, who
will take into account factors including insulin level, blood glucose
levels, the physical condition of the patient, the patient's pulmonary
status, or the like.

[0401]According to the invention, insulin derivative of this invention may
be delivered by inhalation to achieve prolonged duration of action.
Administration by inhalation can result in pharmacokinetics comparable to
subcutaneous administration of insulins. Different inhalation devices
typically provide similar pharmacokinetics when similar particle sizes
and similar levels of lung deposition are compared.

[0402]According to the invention, insulin derivative of this invention may
be delivered by any of a variety of inhalation devices known in the art
for administration of a therapeutic agent by inhalation. These devices
include metered dose inhalers, nebulizers, dry powder generators,
sprayers, and the like. Preferably, insulin derivative of this invention
is delivered by a dry powder inhaler or a sprayer. There are a several
desirable features of an inhalation device for administering insulin
derivative of this invention. For example, delivery by the inhalation
device is advantageously reliable, reproducible, and accurate. The
inhalation device should deliver small particles, for example, less than
about 10 μm, for example about 1-5 μm, for good respirability. Some
specific examples of commercially available inhalation devices suitable
for the practice of this invention are Turbohaler® (Astra),
Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros® inhaler (Dura),
devices marketed by Inhale Therapeutics, AERx® (Aradigm), the
Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer
(Marquest Medical Products), the Ventolin® metered dose inhaler
(Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.

[0403]As those skilled in the art will recognize, the formulation of
insulin derivative of this invention, the quantity of the formulation
delivered, and the duration of administration of a single dose depend on
the type of inhalation device employed. For some aerosol delivery
systems, such as nebulizers, the frequency of administration and length
of time for which the system is activated will depend mainly on the
concentration of insulin conjugate in the aerosol. For example, shorter
periods of administration can be used at higher concentrations of insulin
conjugate in the nebulizer solution. Devices such as metered dose
inhalers can produce higher aerosol concentrations, and can be operated
for shorter periods to deliver the desired amount of insulin conjugate.
Devices such as powder inhalers deliver active agent until a given charge
of agent is expelled from the device. In this type of inhaler, the amount
of insulin derivative of this invention in a given quantity of the powder
determines the dose delivered in a single administration.

[0404]The particle size of insulin derivative of this invention in the
formulation delivered by the inhalation device is critical with respect
to the ability of insulin to make it into the lungs, and preferably into
the lower airways or alveoli. Preferably, the insulin derivative of this
invention is formulated so that at least about 10% of the insulin
conjugate delivered is deposited in the lung, preferably about 10 to
about 20%, or more. It is known that the maximum efficiency of pulmonary
deposition for mouth breathing humans is obtained with particle sizes of
about 2 μm to about 3 μm. When particle sizes are above about 5
μm pulmonary deposition decreases substantially. Particle sizes below
about 1 μm cause pulmonary deposition to decrease, and it becomes
difficult to deliver particles with sufficient mass to be therapeutically
effective. Thus, particles of the insulin derivative delivered by
inhalation have a particle size preferably less than about 10 μm, more
preferably in the range of about 1 μm to about 5 μm. The
formulation of the insulin derivative is selected to yield the desired
particle size in the chosen inhalation device.

[0405]Advantageously for administration as a dry powder, an insulin
derivative of this invention is prepared in a particulate form with a
particle size of less than about 10 μm, preferably about 1 to about 5
μm. The preferred particle size is effective for delivery to the
alveoli of the patient's lung. Preferably, the dry powder is largely
composed of particles produced so that a majority of the particles have a
size in the desired range. Advantageously, at least about 50% of the dry
powder is made of particles having a diameter less than about 10 μm.
Such formulations can be achieved by spray drying, milling, or critical
point condensation of a solution containing insulin conjugate and other
desired ingredients. Other methods also suitable for generating particles
useful in the current invention are known in the art.

[0406]The particles are usually separated from a dry powder formulation in
a container and then transported into the lung of a patient via a carrier
air stream. Typically, in current dry powder inhalers, the force for
breaking up the solid is provided solely by the patient's inhalation. In
another type of inhaler, air flow generated by the patient's inhalation
activates an impeller motor which deagglomerates the particles.

[0407]Formulations of insulin derivatives of this invention for
administration from a dry powder inhaler typically include a finely
divided dry powder containing the derivative, but the powder can also
include a bulking agent, carrier, excipient, another additive, or the
like. Additives can be included in a dry powder formulation of insulin
conjugate, for example, to dilute the powder as required for delivery
from the particular powder inhaler, to facilitate processing of the
formulation, to provide advantageous powder properties to the
formulation, to facilitate dispersion of the powder from the inhalation
device, to stabilize the formulation (for example, antioxidants or
buffers), to provide taste to the formulation, or the like.
Advantageously, the additive does not adversely affect the patient's
airways. The insulin derivative can be mixed with an additive at a
molecular level or the solid formulation can include particles of the
insulin conjugate mixed with or coated on particles of the additive.
Typical additives include mono-, di-, and polysaccharides; sugar alcohols
and other polyols, such as, for example, lactose, glucose, raffinose,
melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or
combinations thereof; surfactants, such as sorbitols, diphosphatidyl
choline, or lecithin; or the like. Typically an additive, such as a
bulking agent, is present in an amount effective for a purpose described
above, often at about 50% to about 90% by weight of the formulation.
Additional agents known in the art for formulation of a protein such as
insulin analogue protein can also be included in the formulation.

[0408]A spray including the insulin derivatives of this invention can be
produced by forcing a suspension or solution of insulin conjugate through
a nozzle under pressure. The nozzle size and configuration, the applied
pressure, and the liquid feed rate can be chosen to achieve the desired
output and particle size. An electrospray can be produced, for example,
by an electric field in connection with a capillary or nozzle feed.
Advantageously, particles of insulin conjugate delivered by a sprayer
have a particle size less than about 10 μm, preferably in the range of
about 1 μm to about 5 μm.

[0409]Formulations of insulin derivatives of this invention suitable for
use with a sprayer will typically include the insulin derivative in an
aqueous solution at a concentration of about 1 mg to about 20 mg of
insulin conjugate per ml of solution. The formulation can include agents
such as an excipient, a buffer, an isotonicity agent, a preservative, a
surfactant, and, preferably, zinc. The formulation can also include an
excipient or agent for stabilization of the insulin derivative, such as a
buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk
proteins useful in formulating insulin conjugates include albumin,
protamine, or the like. Typical carbohydrates useful in formulating
insulin conjugates include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The insulin derivative formulation can also include
a surfactant, which can reduce or prevent surface-induced aggregation of
the insulin conjugate caused by atomization of the solution in forming an
aerosol. Various conventional surfactants can be employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene
sorbitol fatty acid esters. Amounts will generally range between about
0.001 and about 4% by weight of the formulation.

[0410]Pharmaceutical compositions containing an insulin derivative
according to the present invention may also be administered parenterally
to patients in need of such a treatment. Parenteral administration may be
performed by subcutaneous, intramuscular or intravenous injection by
means of a syringe, optionally a pen-like syringe. Alternatively,
parenteral administration can be performed by means of an infusion pump.
Further options are to administer the insulin nasally or pulmonally,
preferably in compositions, powders or liquids, specifically designed for
the purpose.

[0411]Injectable compositions of the insulin derivatives of the invention
can be prepared using the conventional techniques of the pharmaceutical
industry which involve dissolving and mixing the ingredients as
appropriate to give the desired end product. Thus, according to one
procedure, an insulin derivative according to the invention is dissolved
in an amount of water which is somewhat less than the final volume of the
composition to be prepared. An isotonic agent, a preservative and a
buffer is added as required and the pH value of the solution is
adjusted--if necessary--using an acid, e.g. hydrochloric acid, or a base,
e.g. aqueous sodium hydroxide as needed. Finally, the volume of the
solution is adjusted with water to give the desired concentration of the
ingredients.

[0413]In a further aspect of the invention the formulation further
comprises a pharmaceutically acceptable preservative which may be
selected from the group consisting of phenol, o-cresol, m-cresol,
p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,
2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl
alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,
imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl
p-hydroxybenzoate, benzethonium chloride, chlorphenesine
(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a further
aspect of the invention the preservative is present in a concentration
from 0.1 mg/ml to 20 mg/ml. In a further aspect of the invention the
preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In
a further aspect of the invention the preservative is present in a
concentration from 5 mg/ml to 10 mg/ml. In a further aspect of the
invention the preservative is present in a concentration from 10 mg/ml to
20 mg/ml. Each one of these specific preservatives constitutes an
alternative aspect of the invention. The use of a preservative in
pharmaceutical compositions is well-known to the skilled person. For
convenience reference is made to Remington: The Science and Practice of
Pharmacy, 19th edition, 1995.

[0414]In a further aspect of the invention the formulation further
comprises an isotonic agent which may be selected from the group
consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an
amino acid (e.g. L-glycine, L-histidine, arginine, lysine, isoleucine,
aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol
(glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediol,
1,3-butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof.
Any sugar such as mono-, di-, or polysaccharides, or water-soluble
glucans, including for example fructose, glucose, mannose, sorbose,
xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin,
cyclodextrin, soluble starch, hydroxyethyl starch and
carboxymethylcellulose-Na may be used. In one aspect the sugar additive
is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at
least one --OH group and includes, for example, mannitol, sorbitol,
inositol, galactitol, dulcitol, xylitol, and arabitol. In one aspect the
sugar alcohol additive is mannitol. The sugars or sugar alcohols
mentioned above may be used individually or in combination. There is no
fixed limit to the amount used, as long as the sugar or sugar alcohol is
soluble in the liquid preparation and does not adversely effect the
stabilizing effects achieved using the methods of the invention. In one
aspect, the sugar or sugar alcohol concentration is between about 1 mg/ml
and about 150 mg/ml. In a further aspect of the invention the isotonic
agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a
further aspect of the invention the isotonic agent is present in a
concentration from 1 mg/ml to 7 mg/ml. In a further aspect of the
invention the isotonic agent is present in a concentration from 8 mg/ml
to 24 mg/ml. In a further aspect of the invention the isotonic agent is
present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these
specific isotonic agents constitutes an alternative aspect of the
invention. The use of an isotonic agent in pharmaceutical compositions is
well-known to the skilled person. For convenience reference is made to
Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0417]A composition for nasal administration of an insulin derivative
according to the present invention may, for example, be prepared as
described in European Patent No. 272097 (to Novo Nordisk A/S).

[0418]Compositions containing insulin derivatives of this invention can be
used in the treatment of states which are sensitive to insulin. Thus,
they can be used in the treatment of type 1 diabetes, type 2 diabetes and
hyperglycaemia for example as sometimes seen in seriously injured persons
and persons who have undergone major surgery. The optimal dose level for
any patient will depend on a variety of factors including the efficacy of
the specific insulin derivative employed, the age, body weight, physical
activity, and diet of the patient, on a possible combination with other
drugs, and on the severity of the state to be treated. It is recommended
that the daily dosage of the insulin derivative of this invention be
determined for each individual patient by those skilled in the art in a
similar way as for known insulin compositions.

[0419]Where expedient, the insulin derivatives of this invention may be
used in mixture with other types of insulin, e.g. insulin analogues with
a more rapid onset of action. Examples of such insulin analogues are
described e.g. in the European patent applications having the publication
Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo Nordisk A/S) and EP
383472 (Eli Lilly & Co.).

[0420]In a further aspect of the present invention the present compounds
are administered in combination with one or more further active
substances in any suitable ratios. Such further active agents may be
selected from antidiabetic agents, antihyperlipidemic agents, antiobesity
agents, antihypertensive agents and agents for the treatment of
complications resulting from or associated with diabetes.

[0421]Suitable antidiabetic agents include insulin, GLP-1 (glucagon like
peptide-1) derivatives such as those disclosed in WO 98/08871 (Novo
Nordisk A/S), which is incorporated herein by reference, as well as
orally active hypoglycemic agents.

[0424]With "B(1-29)" is meant a natural insulin B chain or an analogue
thereof lacking the B30 amino acid residue. "A(1-21)" means the natural
insulin A chain or an analogue thereof.

[0425]With "B1", "A1" etc. is meant the amino acid residue in position 1
in the B chain of insulin (counted from the N-terminal end) and the amino
acid residue in position 1 in the A chain of insulin (counted from the
N-terminal end), respectively. The amino acid residue in a specific
position may also be denoted as e.g. PheB1 which means that the
amino acid residue in position B1 is a phenylalanine residue.

[0426]With "Insulin" as used herein is meant human insulin with disulfide
bridges between CysA7 and CysB7 and between CysA20 and
CysB19 and an internal disulfide bridge between CysA6 and
CysA11, porcine insulin and bovine insulin.

[0427]By "insulin analogue" as used herein is meant a polypeptide which
has a molecular structure which formally can be derived from the
structure of a naturally occurring insulin, for example that of human
insulin, by deleting and/or substituting at least one amino acid residue
occurring in the natural insulin and/or by adding at least one amino acid
residue. The added and/or substituted amino acid residues can either be
codable amino acid residues or other naturally occurring amino acid
residues or purely synthetic amino acid residues.

[0428]The insulin analogues may be such wherein position 28 of the B chain
may be modified from the natural Pro residue to one of Asp, Lys, or Ile.
In another aspect Lys at position B29 is modified to Pro. In one aspect
B30 may be Lys and then B29 can be any codable amino acid except Cys,
Met, Arg and Lys. Also, Asn at position A21 may be modified to Ala, Gln,
Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to
Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position
B3 may be modified to Lys or Asp. Further examples of insulin analogues
are desB30 human insulin, desB30 human insulin analogues; insulin
analogues wherein one or both of B1 and B2 have been deleted; insulin
analogues wherein the A-chain and/or the B-chain have an N-terminal
extension and insulin analogues wherein the A-chain and/or the B-chain
have a C-terminal extension. Further insulin analogues are such wherein.
Thus one or two Arg may be added to position B1. Also one or more of
B26-B30 may have been deleted

[0429]By "insulin derivative" as used herein is meant a naturally
occurring insulin or an insulin analogue which has been chemically
modified, e.g. by introducing a side chain in one or more positions of
the insulin backbone or by oxidizing or reducing groups of the amino acid
residues in the insulin or by converting a free carboxylic group to an
ester group or acylating a free amino group or a hydroxy group.

[0430]The expression "a codable amino acid" or "a codable amino acid
residue" is used to indicate an amino acid or amino acid residue which
can be coded for by a triplet ("codon") of nucleotides.

[0431]α-Asp is the L-form of --HNCH(CO--)CH2COOH.

[0432]β-Asp is the L-form of --HNCH(COOH)CH2CO--.

[0433]α-Glu is the L-form of --HNCH(CO--)CH2CH2COOH.

[0434]γ-Glu is the L-form of --HNCH(COOH)CH2CH2CO--.

[0435]The expression "an amino acid residue having a carboxylic acid group
in the side chain" designates amino acid residues like Asp, Glu and hGlu.
The amino acids can be in either the L- or D-configuration. If nothing is
specified it is understood that the amino acid residue is in the L
configuration.

[0437]When an insulin derivative according to the invention is stated to
be "soluble at physiological pH values" it means that the insulin
derivative can be used for preparing insulin compositions that are fully
dissolved at physiological pH values. Such favourable solubility may
either be due to the inherent properties of the insulin derivative alone
or a result of a favourable interaction between the insulin derivative
and one or more ingredients contained in the vehicle.

[0438]The following abbreviations have been used in the specification and
examples:

[0464]All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference in their
entirety and to the same extent as if each reference were individually
and specifically indicated to be incorporated by reference and were set
forth in its entirety herein (to the maximum extent permitted by law).

[0465]All headings and sub-headings are used herein for convenience only
and should not be construed as limiting the invention in any way.

[0466]The use of any and all examples, or exemplary language (e.g., "such
as") provided herein, is intended merely to better illuminate the
invention and does not pose a limitation on the scope of the invention
unless otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.

[0467]The citation and incorporation of patent documents herein is done
for convenience only and does not reflect any view of the validity,
patentability, and/or enforceability of such patent documents.

[0468]This invention includes all modifications and equivalents of the
subject matter recited in the claims appended hereto as permitted by
applicable law.

Examples

[0469]The following examples and general procedures refer to intermediate
compounds and final products identified in the specification and in the
synthesis schemes. The preparation of the compounds of the present
invention is described in detail using the following examples, but the
chemical reactions described are disclosed in terms of their general
applicability to the preparation of compounds of the invention.
Occasionally, the reaction may not be applicable as described to each
compound included within the disclosed scope of the invention. The
compounds for which this occurs will be readily recognised by those
skilled in the art. In these cases the reactions can be successfully
performed by conventional modifications known to those skilled in the
art, that is, by appropriate protection of interfering groups, by
changing to other conventional reagents, or by routine modification of
reaction conditions. Alternatively, other reactions disclosed herein or
otherwise conventional will be applicable to the preparation of the
corresponding compounds of the invention. In all preparative methods, all
starting materials are known or may easily be prepared from known
starting materials. All temperatures are set forth in degrees Celsius and
unless otherwise indicated, all parts and percentages are by weight when
referring to yields and all parts are by volume when referring to
solvents and eluents.

[0470]The compounds of the invention can be purified by employing one or
more of the following procedures which are typical within the art. These
procedures can--if needed--be modified with regard to gradients, pH,
salts, concentrations, flow, columns and so forth. Depending on factors
such as impurity profile, solubility of the insulins in question
etcetera, these modifications can readily be recognised and made by a
person skilled in the art.

[0471]After acidic HPLC or desalting, the compounds are isolated by
lyophilisation of the pure fractions.

[0472]After neutral HPLC or anion exchange chromatography, the compounds
are desalted, precipitated at isoelectrical pH, or purified by acidic
HPLC.

[0473]Typical Purification Procedures:

[0474]The HPLC system is a Gilson system consisting of the following:
Model 215 Liquid handler, Model 322-H2 Pump and a Model 155 UV Dector.
Detection is typically at 210 nm and 280 nm.

[0503]Pumps and detectors are controlled by MassChrom 1.1.1 software
running on a Macintosh G3 computer. Gilson Unipoint Version 1.90 controls
the auto-injector.

[0504]The HPLC pump is connected to two eluent reservoirs containing:

[0505]A: 0.01% TFA in water

[0506]B: 0.01% TFA in acetonitrile

[0507]The analysis is performed at room temperature by injecting an
appropriate volume of the sample (preferably 10 μl) onto the column,
which is eluted, with a gradient of acetonitrile. The eluate from the
column passed through the UV detector to meet a flow splitter, which
passed approximately 30 μl/min (1/50) through to the API Turbo
ion-spray interface of API 3000 spectrometer. The remaining 1.48 ml/min
(49/50) is passed through to the ELS detector.

[0508]The HPLC conditions, detector settings and mass spectrometer
settings used are giving in the following table.

[0521]After the DAD the flow was divided yielding approx 1 ml/min to the
ELS and 0.5 ml/min to the MS.

[0522]MALDI-TOF-MS spectra were recorded on a Bruker Autoflex II TOF/TOF
operating in linear mode using a matrix of sinnapinic acid, a nitrogen
laser and positive ion detection. Accelerating voltage: 20 kV.

[0527]Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250
ml) and the mixture was heated to reflux. N,N-dimethylformamide
di-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4 hours.
The mixture was refluxed overnight. The solvent was removed in vacuo at
50° C., and the crude material was suspended in DCM/AcOEt (500 ml,
1:1) and stirred for 15 mins. The solids were collected by filtration and
triturated with DCM (200 ml). The filtrated were evaporated in vacuo to
give crude mono-tert-butyl hexadecandioate, 30 grams. This material was
suspended in DCM (50 ml), cooled with ice for 10 mins, and filtered. The
solvent was removed in vacuo to leave 25 gram crude mono-tert-butyl
hexadecandioate, which was recrystallized from heptane (200 ml) to give
mono-tert-butyl hexadecandioate, 15.9 g (33%). Alternatively to
recrystallization, the mono-ester can be purified by silica
chromatography in AcOEt/heptane.

[0531]Succinimidyl tert-butyl hexadecandioate (1 g, 2.27 mmol) was
dissolved in DMF (15 ml) and treated with L-Glu-OtBu (0.51 g, 2.5 mmol)
and DIEA (0.58 ml, 3.41 mmol) and the mixture was stirred overnight. The
solvent was evaporated in vacuo, and the crude product was dissolved in
AcOEt, and washed twice with 0.2M HCl, with water and brine. Drying over
MgSO4 and evaporation in vacuo gave w-tert-butyl
carboxy-pentadecanoyl-L-glutamyl-α-tert-butyl ester, 1.2 g (100%).

[0540]This compound was prepared in analogy with example 1 via reaction of
L-GluOtBu with tert-butyl succinimidyl octadecandioate followed by
activation with TSTU, activation with TSTU, reaction with
3-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid
activation with TSTU, coupling with DesB30 human insulin and deprotection
by TFA.

[0552]2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonylpentadecanoylamino)etho-
xy]-ethoxy}ethoxy)ethoxy]propionylamino}pentanedioic acid 5-benzyl ester
1-tert-butyl ester (0.2 g, 0.23 mmol) was dissolved in THF. The flask was
filled with N2, and palladium (0.3 g, 10% on carbon, 50% water) was
added, and the flask was equipped with a balloon filled with H2. The
mixture was stirred for 16 h at rt, and filtered through celite, washing
with THF. The filtrate was concentrated to yield an oil (0.16 g, 89%).

[0558]2-{3-[2-(2-{2-[2-(ω-tert-Butoxycarbonyl-pentadecanoylamino)eth-
oxy]-ethoxy}ethoxy)ethoxy]-propionylamino}pentanedioic acid
α-tert-butyl ester 1-(2,5-dioxopyrrolidin-1-yl)ester was coupled to
desB30 insulin in similar fashion as described in Example 1 The
intermediate product was purified by preparative HPLC (C18-5 cm
dia.) before treating with TFA. The final product was purified by
preparative HPLC (C4, 2 cm dia.) then (C4, 1 cm dia.) (20-60%
acetonitrile).

[0563]This compound was prepared in analogy with example 1 via reaction of
H2N(CH2CH2O)4CH2CH2COOtBu (Quanta
Biodesign, OH, USA) with mono-succinimidyl oc-tadecandioate followed by
activation with TSTU, reaction with L-Glu-OtBu, activation with TSTU,
coupling with DesB30 human insulin and deprotection by TFA.

[0566]The compound was prepared in the same manner as with
N.sup.εB29-3-[2-(2-{2-[2-(ω-carboxy-pentadecanoylamino)-eth-
oxy]-ethoxy}-ethoxy)-ethoxy]-propionyl-γ-glutamyl desB30 insulin
using octadecanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl
ester as the starting material.

[0583]This compound was prepared by deprotection of
N-(3-{2-[2-(3-tert-butoxycarbonylaminopropoxy)-ethoxy]ethoxy}propyl)succi-
namic acid (1.56 mmol) by means of TFA, followed by reaction with
octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (1.56
mmol). as described in example 8 step3.

[0603]The mixture was stirred under nitrogen overnight, evaporated to
dryness, dissolved in EtAc (50 mL) which subsequently was extracted 2
times with HCL (0.1 M). The organic phase was dried with MgSO4,
filtered and evaporated resulting in an slightly yellow oil (5 g,
containing small amounts of solvent)

[0608]pH was adjusted to 8.5 with DIPEA and the mixture was stirred
overnight under nitrogen. The mixture was subsequently evaporated to
dryness and redissolved in EtAc (50 mL). The EtAc phase was extracted
×3 with HCl (0.1 M), the organic layer dried over magnesium
sulphate, filtered and evaporated resulting in a slightly yellow
crystalline oil (6.5 g, content of solvent residues)

[0609]LCMS (Method 6): Rt 4.31 min; m/z (M+1) 517. Calcd: 517.

[0610]The crude product was used for further reaction without further
purification.

[0616]This was dissolved in THF (30 mL), pH was adjusted to 8.2 with DIPEA
(0.4 mL) and H-glu-OtBu (1.7 g, 4.9 mmol) was added together with DMF (10
mL). The mixture was stirred at RT for 3 h, filtration followed by
evaporation afforded a thick yellow oil.

[0629]The product was treated with TFA/DCM 1/1 (20 mL) by stirring at RT
for 1 h, subsequent evaporation to dryness and stripping with DCM 40
mL×2 resulted in the deprotected product which was dissolved in
water and freeze dried giving 540 mg of the wanted product.

[0678]7-(2-{2-[2-(3-Carboxy-propionylamino)-ethoxy]-ethoxy}-ethylcarbamoyl-
)-heptanoic acid tert-butyl ester (1.3 g, 2.83 mmol) was activated with
TSTU and subsequently the crude product was reacted with H-glu-OtBu (0.86
g, 4.2 mmol). After work up using the method described in example 8, the
product was further purified on Gilson using acidic HPLC on a C18 column
(Jones, Kromasil RP18 5 μm 15×225 mm).

[0685]This compound was prepared similarly as described in example 4. The
intermediate
15-[2-(2-{2-[2-(2-Carboxyethoxy)ethoxy]ethoxy}ethoxy)ethylcarbamoyl]penta-
decanoic acid tert-butyl ester was activated to the OSu-ester using TSTU
and coupled to desB30 human insulin. Deprotection using TFA afforded the
title compound.

[0729]The affinity of the insulin analogues of the invention for the human
insulin receptor was determined by a SPA assay (Scintillation Proximity
Assay) microtiterplate antibody capture assay. SPA-PVT antibody-binding
beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) were
mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium
chloride, 10 mM MgSO4, 0.025% Tween-20). Reagent mix for a single
Packard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a
1:5000 diluted purified recombinant human insulin receptor-exon 11, an
amount of a stock solution of A14 Tyr[125I]-human insulin
corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a
1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to
a total of 12 ml. A total of 100 μl was then added and a dilution
series is made from appropriate samples. To the dilution series was then
added 100 μl of reagent mix and the samples were incubated for 16
hours while gently shaken. The phases were the then separated by
centrifugation for 1 min and the plates counted in a Topcounter. The
binding data were fitted using the nonlinear regression algorithm in the
GraphPad Prism 2.01 (GraphPad Software, San Diego, Calif.).

[0731]The test substance will be dosed pulmonary by the drop instillation
method. In brief, male Wistar rats (app.250 g) are anaesthesized in app.
60 ml fentanyl/dehydrodenzperidol/dormicum given as a 6.6 ml/kg sc
primingdose and followed by 3 maintainance doses of 3.3 ml/kg sc with an
interval of 30 min. Ten minutes after the induction of anaesthesia, basal
samples are obtained from the tail vein (t=-20 min) followed by a basal
sample immediately prior to the dosing of test substance (t=0). At t=0,
the test substance is dosed intra tracheally into one lung. A special
cannula with rounded ending is mounted on a syringe containing the 200 ul
air and test substance (1 ml/kg). Via the orifice, the cannula is
introduced into the trachea and is forwarded into one of the main
bronchi--just passing the bifurcature. During the insertion, the neck is
palpated from the exterior to assure intratracheal positioning. The
content of the syringe is injected followed by 2 sec pause. Thereafter,
the cannula is slowly drawn back. The rats are kept anaesthesized during
the test (blood samples for up to 4 hrs) and are euthanized after the
experiment.